Outline

We have previously shown that angiotensinogen, like other members of the serine protease inhibitor (serpins) family has anti-angiogenic properties in vitro on cultured endothelial cells and in ovo in the chick chorioallantoic membrane assay. By staining blood vessels of histological sections of the chick-chorioallantoic membrane with an endothelial-specific cell marker lectin we quantified vessel density in angiotensinogen treated areas and control areas: 48 hours after angiotensinogen was locally applied on the chick-chorioallantoic membrane we observed a 70% decrease in mesodermic vessel density in comparison to the control sections. Human angiotensinogen transfected chinese-hamster-ovary-cells grafted onto the surface of the chick-chorioallantoic membrane for 48 hours and constantly producing and secreting human angiotensinogen are leading to a similar decrease in vessel density of mesodermal blood vessels in comparison to non-transfected cells. As human angiotensinogen does not interact enzymatically with the renin-angiotensin-system of the chicken embryo, this anti-angiogenic effect is directly due to angiotensinogen. As assessed by the incorporation of bromo-deoxyuridine we observed a strong decrease in endothelial cell proliferation in the chick-chorioallantoic membrane vasculature of angiotensinogen treated areas in comparison to controls. Two days after local angiotensinogen treatment was initiated, increased apoptosis of endothelial cells of mesodermal blood vessels was detected by transferase-mediated deoxyuridine triphosphate nick end labeling assay. As assessed by in situ hybridization the expression pattern of the main vascular growth factor genes were not altered by angiotensinogen treatment. Angiotensinogen can therefore impair angiogenesis without altering the expression level of vascular growth factors through induction of apoptosis and decreased proliferation of endothelial cells.